SERS‐based immunomagnetic bead for rapid detection of H5N1 influenza virus

Abstract The surface‐enhanced Raman scattering (SERS) has recently drawn attention in the detection of respiratory viruses, but there have been few reports of the direct detection of viruses. In this study, a sandwich immunomagnetic bead SERS was established for the rapid diagnosis of the H5N1 influenza virus. The detection limit was estimated to be 5.0 × 10−6 TCID50/ml. The method showed excellent specificity with no cross‐reaction with H1N1, H5N6 or H9N2. The H5N1 influenza virus detection accuracy of the SERS method was 100% in chicken embryos. The results hold great promise for the utilization of SERS as an innovative approach in the diagnosis of influenza virus.


| INTRODUCTION
The influenza A virus of the H5N1 subtype is a highly pathogenic avian influenza (HPAI) that leads to severe acute respiratory infection in humans, with a fatality rate of more than 50%. 1 An efficient analytical method capable of the reliable detection and identification of viruses is necessary for the adequate management of epidemic. Accurate analytical techniques or rapid detection assays are used for virus identification or quantification. The commonly used methods to detect or quantify viruses can be subdivided into virus culture, serological test, nucleic acid-based detection methods and biosensors-based point-of-care testing system. 2 The virus culture and reverse transcription PCR (RT-PCR) had been the gold standard lab methods for the H5N1 influenza virus detection. 3 Real-time PCR presents high specificity, sensitivity, stability and experimental reliability and is currently used to detect influenza viruses and other pathogens. However, complex processes and long sample preparation times are required of these detection methods.
Surface-enhanced Raman scattering (SERS) as a molecular fingerprint spectrum has been applied to a variety of influenza A virus subtypes, such as the H1N1, H7N9, H3N2, H5N1 and the novel coronavirus SARS-CoV-2. 4 In the present study, we established a highly sensitive and label-free SERS detection method for tracking the Raman signal of the H5N1 influenza virus (Figure 1). The H5N1 influenza virus was shown to bond with a biotinylated primary antibody on the magnetic beads and later was combined with a secondary antibody to form an immunomagnetic beads sandwich immunocomplexes (IMBSIs). The strong SERS signal of the H5N1 influenza virus can be sensitively detected through the in-situ reduction of nano-silver serving as a SERS substrate.

| Virus and Raman instrument
Avian influenza viruses (A/chicken/Jilin/9/2004/H5N1) were obtained from our laboratory stock. The H5N1 influenza virus titer was 5.0 Â 10 6 TCID 50 /ml (50% tissue culture infective dose), as determined by the monitored cytopathic effects (CPEs) of the infected cells according to standard procedures. 5 The influenza viruses strains

| Preparation of sandwich immunomagnetic beads
The times to remove unbound viruses. Then, 0.5 μg/ml of detection anti-H5N1 hemagglutinin monoclonal antibody (Sino Biological Inc., Beijing, China) was added for 30 min at room temperature.
Finally, the conjugated IMBs sandwich immunocomplexes (IMBSIs) were pipetted for washing in PBS and subsequent magnetic separation.

| In situ reduction of nano-silver and TEM detection
The in situ reduction of nano-silver was used for synthesis according to our previously study. 6 Briefly, 50 μl of IMBSIs was centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. Then, 50 μl (1.72 mg/ml) of AgNO 3 was mixed with 50 μl of IMBSIs for 5 min.
Next, 50 μl of a 0.378 mg/ml NaBH 4 solution was rapidly added, vortexed and centrifuged, and the supernatant was discarded. The obtained samples were stored at 4 C and were stable for months without light. Microstructures of the magnetic beads and IMBSIs in the in-situ reduction of nano-silver (IMBSIs@Ag) were investigated by transmission electron microscopy (TEM).

| SERS measurements of H5N1 influenza virus
The SERS measurements were performed in different samples, 3 | RESULTS

| Working principle of the IMBSIs@Ag-SERS method
In this study, we used IMBs composed of antibodies as molecular recognition elements to capture the H5N1 influenza virus, and then the second antibody was added to the IMBs to form sandwich immunocomplexes. This structure is referred to as IMBSIs for the in-situ reduction for SERS of silver nanoparticles. The Raman spectra of IMBSIs@Ag-SERS+H5N1 showed a prominent peak at 1053 cm À1 compared to the unenhanced IMBSIs+H5N1 and the enhanced IMBSIs@Ag-SERS or IMBs@Ag-SERS (Figure 2A). It showed excellent capturing efficiency at 0.5 μg/ml of the primary antibody concentration ( Figure 2B). It can be clearly observed from the TEM image that nano-silver was synthesized around the surface of IMBSIs ( Figure 2C, D), which resulted in the specific amplified signal. The results display that the Raman peak could be used for quantitative determination of viruses using the example of influenza A virus.

| Specificity, uniformity and repeatability of SERS assay
The result showed a consistent Raman intensity at 1053 cm À1 in three independent H5N1 influenza virus samples, which were performed again in triplicate ( Figure 3A) Figure 3B).
The H5N1 influenza virus was 10-fold diluted from 5.0 Â 10 6 to 5.0 Â 10 À7 TCID 50 /ml. As shown in Figure 4A, the limit of detection (LOD) is 5.0 Â 10 À6 TCID 50 /ml in Raman spectra. The Raman peak height reduced when the H5N1 influenza virus concentration decreased, and the calibration showed a linear dependence between the virus concentration and the Raman peak height at 1053 cm À1 (R 2 = 0.9566) ( Figure 4B).

| Identification of H5N1 influenza virus from chicken embryos
To demonstrate the applicability of the SERS in IMBSIs@Ag in allantois fluid with the H5N1 influenza virus infected and uninfected.
The results were consistent with Raman spectra and HA tests. Twelve samples were randomly selected from 200 infected and 200 uninfected chicken embryos. Nine of these samples tested positive, and three samples tested negative for the H5N1 influenza virus in the HA test (data aren't shown). The Raman spectra results showed a peak at 1053 cm À1 from nine samples but no peak for three of the samples from twelve random samples ( Figure 4C). The detection accuracy of Raman spectroscopy using the IMBSIs sandwich method was 100%.

| DISCUSSION
A wide variety of SERS-based schemes were developed to detect viruses due to the different types of signal-enhancing substrates. 7 Most of them consumed antisense oligonucleotides to capture the genome of the virus. 8  surface of the nanoparticles to serve as Raman reporters. 9 We have developed a label-free SERS detection of the H5N1 influenza virus.
Depending on the characteristics of antibody-virus-antibody interactions, the H5N1 influenza virus was captured with primary antibody in immunomagnetic beads. The secondary antibody was added, followed by the in situ reduction of silver nanoparticles. Interestingly, the IMBs captured H5N1 and showed a narrow peak at 1053 cm À1 without the presence of the second antibody, but had a lower sensitivity than the IMBSIs captured H5N1. There was a gap between the sample and nano-silver, which enhanced the SERS effect. The gap width was the distance of the second antibody that perhaps produced the electromagnetic enhancement in the Raman signal. Influenza hemagglutinin (HA) protein is a glycoprotein found on the surface of influenza viruses. Therefore, we attempted to replace influenza virus H5N1 with H5N1 HA protein in IMBs. The data show that there is still an obvious Raman peak at 1053 cm À1 . Some Raman peaks at different Raman shift can be observed in HA protein without SERS (Figure 2A).
The IMBs sandwich immunocomplexes enhanced the peak intensity of H5N1 at 1053 cm À1 compared to the rest of the spectrum.
In another study, Moon et al. utilized  quantification is based on tracking the Raman signal from the reporter molecule 4-MBA instead of the Raman signal from H5N1 influenza virus. Our study has higher sensitivity and a much lower detection limit (LOD = 5.0 Â 10 À6 TCID 50 /ml). The result also confirms our conjecture that AgNP grows on the secondary antibodies but not on the primary antibodies, leading to the outermost components of organisms being enhanced. Raman reporters can be detected rapidly and conveniently, but they are easy to lose or be quenched. The ultrasensitive sandwich method directly detected the sample, but not the Raman reporters. The Raman peaks were generated from enhanced sandwich immunocomplexes but not from the immunomagnetic beads.
The SERS method has been widely used in biological detection for its fast, accurate and fingerprint characteristics. Some researchers have shown the detection of bacteria by SERS spectra directly. 13 Viruses differ from bacteria in that they depend on host cells for survival, replication and propagation, and it is difficult to acquire pure viruses with no impurities (contents and medium of host cells) like bacteria. Some researchers have quantitatively assayed these samples by analysing the peak height or peak area ratios, but the major quantitative analysis is based on the enhanced signals in the material (noble metals and compounds) or method (self-assembled monolayer layers and SERS tags [14][15][16] ). Although the result showed a linear relationship between the peak height and the virus concentration, the virus was still not able to be quantified accurately. The quantitative detection of virus was exploratory in the sensitive detection of the H5N1 influenza virus in this study. It was essential for improving the quantitative ability and widespread application in the future. 17 This label-free sandwich method demonstrates a real applicability in using SERS for real clinical samples. The method with high enhanced ability, stability, specificity and susceptibility will be critical for the application of SERS in detection of viruses. In the future, this in-situ Label-Free SERS method holds great potential for applications in portable and rapid